Method of modulating the activity of a nucleic acid molecule

ABSTRACT

The present invention relates, in general, to agents that modulate the pharmacological activity of nucleic acid molecules and, in particular, to agents that bind therapeutic or diagnostic nucleic acid molecules in a sequence independent manner and modulate (e.g., inhibit or reverse) their activity. The invention also relates to compositions comprising such agents and to methods of using same.

This application is a continuation-in-part of International ApplicationNo. PCT/US2008/004119, filed Mar. 31, 2008, which claims the benefit ofU.S. Provisional Application No. 60/920,807, filed Mar. 30, 2007. Thisapplication also claims priority from U.S. Provisional Application No.61/243,078, filed Sep. 16, 2009. The entire contents of InternationalApplication No. PCT/US2008/004119, U.S. Provisional Application No.60/920,807 and U.S. Provisional Application No. 61/243,078 are herebyincorporated by reference.

This invention was made with government support under Grant No. RO1HL65222 awarded by the National Institutes of Health. The government hascertain rights in the invention.

TECHNICAL FIELD

The present invention relates, in general, to agents that modulate thefunctional activity of nucleic acid molecules and, in particular, toagents that bind therapeutic and/or diagnostic nucleic acid molecules ina sequence independent manner and modulate (e.g., inhibit or reverse)their activity. The invention also relates to compositions comprisingsuch agents and to methods of using same.

BACKGROUND

Aptamers are single-stranded nucleic acid (DNA or RNA) ligands thatpossess a number of features that render them useful as therapeuticagents. They are relatively small (8 kDa to 15 kDa) synthetic compoundsthat possess high affinity and specificity for their target molecules(equilibrium dissociation constants ranging from, for example, 0.05-1000nM). Thus, they embody the affinity properties of monoclonal antibodiesand single chain antibodies (scFv's) with the chemical productionproperties of small peptides. While initial studies demonstrated the invitro use of aptamers for studying protein function, more recent studieshave demonstrated the utility of these compounds for studying in vivoprotein function (Floege et al, Am J Pathol 154:169-179 (1999),Ostendorf et al, J Clin Invest 104:913-923 (1999), Dyke, Circulation114(23):2490-7 (2006), Group, Retina 22(2):143-52 (2002), Group,Opthalmology 110(5):979-86 (2003), Nimjee et al, Mol. Ther. 14(3):408-15(2006), Nimjee et al, Trends Cardiovasc Med. 15(1):41-5 (2005), Nimjeeet al, Annu. Rev. Med. 56:555-83 (2005), Rusconi et al, Nat. Biotechnol.22(11):1423-8 (2004)). In addition, animal studies to date have shownthat aptamers and compounds of similar composition are well tolerated,exhibit low or no immunogenicity, and are thus suitable for repeatedadministration as therapeutic compounds (Floege et al, Am J Pathol154:169-179 (1999), Ostendorf et al, J Clin Invest 104:913-923 (1999),Griffin et al, Blood 81:3271-3276 (1993), Hicke et al, J Clin Invest106:923-928 (2000), Dyke, Circulation 114(23):2490-7 (2006), Group,Retina 22(2):143-52 (2002), Group, Opthalmology 110(5):979-86 (2003),Nimjee et al, Mol. Ther. 14(3):408-15 (2006), Nimjee et al, TrendsCardiovasc Med. 15(1):41-5 (2005), Nimjee et al, Annu. Rev. Med.56:555-83 (2005), Rusconi et al, Nat. Biotechnol. 22(11):1423-8 (2004)).

As synthetic compounds, site specific modifications can be made toaptamers to rationally alter their bioavailability and mode ofclearance. For example, it has been found that 2′ fluoropyrimidine-modified aptamers in the 10 kDa to 12 kDa size range have ashort circulating half-life (˜10 minutes) following bolus intravenousadministration but that simple chemical modification of the aptamer orconjugation of the aptamer to a high molecular weight inert carriermolecule (e.g., PEG) increases circulating half-life substantially (6-12hours) (Willis et al, Bioconjug Chem 9:573-582 (1998), Tucker et al, JChromatogr Biomed Sci Appl 732:203-212 (1999), Watson et al, AntisenseNucleic Acid Drug Dev 10:63-75 (2000)). Bioactive and nuclease resistantsingle-stranded nucleic acid ligands comprising L-nucleotides have beendescribed (Williams et al, Proc. Natl. Acad. Sci. 94:11285 (1997); U.S.Pat. No. 5,780,221; Leva et al, Chem. Biol. 9:351 (2002)). These“L-aptamers” are reportedly stable under conditions in which aptamerscomprising nucleotides of natural strandedness (D-nucleotides) (that is,“D-aptamers”) are subject to degradation.

Aptamers can be generated by in vitro screening of complex nucleic-acidbased combinatorial shape libraries (>10¹⁴ shapes per library) employinga process termed SELEX (for Systematic Evolution of Ligands byEXponential Enrichment) (Tuerk et al, Science 249:505-10 (1990)). TheSELEX process consists of iterative rounds of affinity purification andamplification of oligonucleotides from combinatorial libraries to yieldhigh affinity and high specificity ligands. Combinatorial librariesemployed in SELEX can be front-loaded with 2′ modified RNA nucleotides(e.g., 2′ fluoro-pyrimidines) such that the aptamers generated arehighly resistant to nuclease-mediated degradation and amenable toimmediate activity screening in cell culture or bodily fluids. (See alsoU.S. Pat. No. 5,670,637, U.S. Pat. No. 5,696,249, U.S. Pat. No.5,843,653, U.S. Pat. No. 6,110,900, U.S. Pat. No. 5,686,242, U.S. Pat.No. 5,475,096, U.S. Pat. No. 5,270,163 and WO 91/19813.)

Over the past decade, the SELEX technology has enabled the generation ofhigh affinity and high specificity antagonists to a myriad of proteinsincluding reverse transcriptases, proteases, cell adhesion molecules,infectious viral particles and growth factors (see Gold et al, Annu RevBiochem 64:763-97 (1995)). In particular, this technology has beenemployed to generate potent antagonists of coagulation factors,including factors VIIa, IXa, Xa and thrombin, transcription factors,autoimmune antibodies, cell surface receptors, as well as Von Willebrandfactor and GPIIb-IIIa (see, for example, Rusconi et al, Thrombosis andHaemostasis 83:841-848 (2000), White et al, J. Clin Invest 106:929-34(2000), Ishizaki et al, Nat Med 2:1386-1389 (1996), Lee et al, NatBiotechnol 15:41-45 (1997), Nimjee et al, Annu. Rev. Med. 56:555-83(2005)). (See also Published U.S. Application No. 20030083294 anddocuments cited therein, which documents are incorporated herein byreference as is Published U.S. Application No. 20030083294.)

It has been shown previously that the activity of aptamers can bereversed by using matched antidote oligonucleotides (Dyke, Circulation114(23):2490-7 (2006), Rusconi et al, Nat Biotechnol. 22(11):1423-8(2004), Rusconi et al, Nature 419(6902):90-4 (2002)); Published U.S.Application No. 20030083294). Joachimi et al (J. Am. Chem. Soc.129:3036-3037 (2007)) have reported that a G-quadruplex-bindingporphyrin can be used to control the anticoagulant activity of aG-quadruplex-containing aptamer (the porphyrin binding to guanine-richmotifs in the quadruplex).

The present invention results from the identification of agents(referred to below as “universal antidotes”) that can bind therapeuticor diagnostic nucleic acid molecules, such as aptamers, siRNAs, etc., ina sequence independent manner and modulate (e.g., inhibit or reverse)their activity. The universality of the antidotes disclosed hereintranslates into significant savings in time and cost from the standpointof drug development. Further, the nature of these antidotes (detailedbelow) is such that the formation of double-stranded RNA helices isavoided and, therefore, the potential inflammatory response associatedtherewith.

SUMMARY OF THE INVENTION

The present invention relates to agents that modulate (e.g., inhibit orreverse) the functional (e.g., pharmacological or diagnostic) activityof nucleic acid molecules (e.g., aptamers (D or L), ribozymes, antisenseRNAs, internalizing RNAs and RNAi (e.g., siRNAs, microRNAs, and shRNAs),including modified forms of such molecules (e.g., 2′F, 2′OMe, 2′B, 2′I,2′NH, etc.)). More specifically, the invention relates to agents thatbind therapeutic or diagnostic nucleic acid molecules in a sequenceindependent manner (that is, in a manner that is independent of thenucleotide sequence of the nucleic acid molecule) and modulate (e.g.,inhibit or reverse) their activity. These agents can be used to controland/or optimize use of nucleic acids molecules in disease states andother medical settings. The invention also relates to compositionscomprising such agents and to methods of using same.

Objects and advantages of the present invention will be clear from thedescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. Protamine reverses Factor IXa aptamer and Factor Xaptamer activity in APTT assay. Effect of protamine (2.5 μg) on clottingtime of human plasma anti-coagulated with an aptamer (“Ch-9.3t”) tohuman factor IXa (FIG. 1A) or with an aptamer (“11f7t”) to human factorXa (FIG. 1B).

FIG. 2. Protamine reverses both aptamers' activity.

FIG. 3. PPA-DPA reverses Von Willebrand R9.3 aptamer activity.

FIGS. 4A-4C. Protamine reverses Factor IXa aptamer in piganticoagulation model.

FIGS. 5A-5E: Structures of aptamers used and protamine mediated reversalof anticoagulant aptamer activity. (FIG. 5A) Primary sequence andpredicted secondary structures of FIXa (9.3t), FXa (11F7T), VWF (9.3),FII (9D-14), VWF (9.14), FVII (7s⁻¹), FX (7K-5) and FIX (9D-6) aptamers.(FIGS. 5B and 5D) Ability of protamine (2.5 μg per 160 μl) to reverseaptamer 9.3t (150 nM) function in pooled normal human plasma measuredusing the APTT assay. (FIG. 5C) Ability of protamine (2.5 μg per 160 μl)to reverse aptamer 11F7T (250 nM) function in pooled normal human plasmameasured using the APTT assay. (FIG. 5E) Ability of protamine (2.5 μgper 160 μl) to reverse the function of aptamers 9.3t and 11F7Tsimultaneously in pooled normal human plasma measured using the APTTassay. Data is plotted as the mean+/−standard error of the mean (SEM)for three independent measurements.

FIGS. 6A-6F: Polymer mediated reversal of anticoagulant aptameractivity. (FIG. 6A) The ability of protamine and 11 different polymers(2.5 μg per 160 μl) to reverse aptamer 9.3t (150 nM) function wasmeasured in pooled normal human plasma using the APTT assay. (FIGS.6B-6E) The ability of CDP (2.5 μg per 160 μl) to reverse (FIG. 6B)aptamer 9.3t function, (FIG. 6C) aptamer 11F7T function, (FIG. 6D)aptamers 9.3t and 11F7T function simultaneously, (FIG. 6E) aptamersR9D-14, 9D-6 and 7K-5 in normal human plasma was measured using the APTTassay. (FIG. 6F) The ability of CDP (2.5 μg per 160 μl) to reverseaptamer 7S-1 function in pooled normal human plasma was measured usingthe PT assay. The data is plotted as the mean+/−SEM for threeindependent measurements.

FIGS. 7A-7C: Polymer mediated reversal of antiplatelet aptamer function.(FIG. 7A) The ability of CDP to reverse VWF aptamer 9.3 function wasmeasured in normal human whole blood using varying concentrations of thepolymer and the PFA-100 assay. The data is plotted as the mean+/−SEM forthree independent measurements. (FIG. 7B) The ability of PPA-DPA (30KDa) to reverse VWF aptamer 9.3 function was measured in normal humanwhole blood using varying concentrations of the polymer and the PFA-100assay. (FIG. 7C) The ability of CDP (10 μg) to reverse the activity ofthe VWF aptamer 9.14 (60 nM) aptamer was measured in the PFA-100 assay.The data is plotted as the mean+/−SEM for three independentmeasurements.

FIGS. 8A-8C: In vivo aptamer and antidote activity. (FIG. 8A) Theanticoagulant activity of the cholesterol modified FIXa aptamer 9.3t(Ch-9.3t) in swine (n=5) was measured using the ACT assay. (FIG. 8B) Theability of protamine to reverse Ch-9.3t function in vivo (n=5) wasassessed using ACT assay. (FIG. 8C) The ability of CDP to reverseCh-9.3t function in vivo (n=5) was assessed using ACT assay. The datashown are plotted as the mean+/−SEM for the duplicate measurements fromeach animal.

FIGS. 9A-9B: In vitro characterization of CDP and aptamer 9.3tinteraction. (FIG. 9A) Gel electrophoresis of CDP/9.3t mixtures. Theability of the aptamer 9.3t, when mixed with various amounts of CDP, tomigrate through a polyacrylamide gel was examined. While the freeaptamer migrates through the gel to give a single band at the expectedposition (lane 2), all aptamer within the CDP-containing samples[CDP-9.3t ratio 0/1 (lane 2), 2/1 (lane 3), 4/1 (lane 4), 6/1 (lane 5),8/1 (lane 6), and 10/1 (lane 7)] remains within the well, indicative ofan inability to migrate through the gel. (FIG. 9B) Dynamic lightscattering (DLS) of a CDP/9.3t mixture. While neither CDP alone nor 9.3taptamer alone show appreciable scattering (average count rate<2 kcps forboth and neither gives an effective diameter within the dynamic range ofthe instrument (2-5000 nm)), a CDP/9.3t mixture (6.7/1 w/w) shows astrong scattering signal (average count rate 591.7 kcps) and a signalwithin the dynamic range of the instrument (448.9 nm). The gelelectrophoresis and DLS data demonstrate an interaction between CDP and9.3t at the concentrations and ratio used in the swine anticoagulationexperiment (FIG. 8).

FIGS. 10A-10F: The vital signs of the animals during the anticoagulationexperiments compared to pre-antidote injection state. (FIG. 10A) Percentchange in temperature of the animal during the experiment. (FIG. 10B)Percent change in respiratory rate of the animal during the experiment.(FIG. 10C) Percent change in heart rate of the animal during theexperiment. (FIG. 10D) Percent change in systolic pressure of the animalduring the experiment. (FIG. 10E) Percent change in diastolic pressureof the animal during the experiment. (FIG. 10F) Percent change in bloodoxygen level of the animal during the experiment. The data shown areplotted as the mean+/−SEM for the duplicate measurements from eachanimal. All data points are done in duplicates for each animal.

FIG. 11: Evidence for association of CDP-im with siRNA in vivo. Gelelectrophoresis was performed on serum samples from mice that received asingle injection of naked, non-chemically-modified siRNA (fourth andfifth lanes from left) or that received consecutive injections of nakedsiRNA followed by a CDP-Im-containing solution administered one (1)minute apart (second and third lanes from left). The first lane containsthe siRNA stock solution for reference. All four serum-containingsamples produced a distinct band slightly below the wells that is theresult of non-specific interaction between the ethidium bromide (usedfor siRNA detection) and serum components. The fourth and fifth lanesshow no evidence for siRNA remaining in the wells and bands thatco-migrate with the siRNA control (first lane), as expected. Incontrast, the second and third lanes show strong siRNA-containing bandsin the wells and no evidence for siRNA migration through the gel. Suchbands in the wells were not seen when the CDP-Im-containing solution wasadministered alone (data not shown) and are indicative of siRNAinteraction with CDP-Im within the mouse bloodstream.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to agents (“universalantidotes”) (UAs) that can modulate the functional (e.g.,pharmacological) activity of nucleic acid molecules (NAMs), includingtherapeutic and/or diagnostic NAMs, independent of the nucleotidesequence of the NAM. The invention further relates to methods ofmodulating (e.g., reversing/inhibiting) the effect of pharmacologicalNAMs by administering such UAs to human or non-human mammals.Additionally, the invention relates to methods of using UAs of theinvention to assess the activity of NAMs.

Pharmacological NAMs include, but are not limited to, aptamers, siRNAs,microRNAs, shRNAs, antisense RNAs, aptamer-siRNA chimeras, mRNAs,ribozymes and antagomirs, that bind a desired target molecule.

Aptamer target molecules include, generally, peptides, proteins,glycoproteins, polysaccharides and nucleic acids, as well as smallmolecular weight (organic) compounds. More specifically, aptamer targetmolecules can include enzymes (e.g., proteases, including factors VIIa,IXa, Xa, XIa, thrombin and protein C) as well as zymogens thereof.Aptamer target molecules can also include hormones, receptors (includingplatelet receptors, e.g., glycoprotein (gp) IIbIIIa, GPIb-IX-V, GPVI,P2Y₁₂, and PARs), adhesion molecules (e.g, Von Willebrand factor andcollagens) metabolites, cofactors (e.g., Tissue Factor, or coagulationfactors Va and VIIIa), transition state analogs, as well as drugs, dyesand toxins. Aptamers can be made using SELEX methodology (see, forexample, U.S. Pat. Nos. 5,270,163, 5,817,785, 5,595,887, 5,496,938,5,475,096, 5,861,254, 5,958,691, 5,962,219, 6,013,443, 6,030,776,6,083,696, 6,110,900, 6,127,119, and 6,147,204, see also Published U.S.Application No. 20030083294 and documents cited therein)). Aptamersspecific for a wide variety of target molecules are presently available(see, for example, Gold et al, Ann. Rev. Biochem. 64:763 (1995), Nimjeeet al, Annu. Rev. Med. 56:555-83 (2005)).

Details of the production of, for example, siRNAs, microRNAs, andantisense RNAs and of methods of using these NAMs in modifying geneexpression are described, for example, in Dorsett and Tuschl, Nat RevDrug Discov. 3(4):318-29 (2004), Fire et al, Nature 391(6669):806-11(1998), Grishok et al, Cell 106(1):23-34 (2001), Lagos-Quintana, et al,Rna 9(2):175-9 (2003), Leaman et al, Cell 121(7):1097-108 (2005),Martinez et al, Cell 110(5):563-74 (2002), Meister et al, Rna10(3):544-50 (2004), Meister and Tuschl, Nature 431 (2006):343-9 (2004),Pfeffer et al, Science 304(5671):734-6 (2004), Tuschl, Nat Biotechnol.20(5):446-8 (2002), and Tuschl and Borkhardt, Mol Interv. 2(3):158-67(2002).

The present invention relates to a method of modulating (e.g., reversingor inhibiting) the activity of a NAM, for example, by altering itsconformation and thus its function and/or by sterically blocking bindingof the NAM to its target molecule. In accordance with the invention, theUA can be contacted with the targeted NAM, for example, under conditionssuch that it binds to the NAM and modifies the interaction between theNAM and its target molecule. The UA can also interfere with the bindingof the NAM to its target molecule through charge interaction.Modification of the interaction between the NAM and its target moleculecan result from, for example, modification of the NAM structure as aresult of binding by the UA. The UA can bind the free NAM and/or the NAMbound to its target molecule.

UAs of the invention include pharmaceutically acceptable member(s) of agroup of positively charged compounds, including proteins, lipids, andnatural and synthetic polymers that can bind NAMs in, for example,biologically fluids.

Proteinaceous UAs of the invention include protamines, a group ofproteins that yield basic amino acids on hydrolysis and that occurcombined with nucleic acid in the sperm of fish, such as salmon.Protamines are soluble in water, are not coagulated by heat, andcomprise arginine, alanine and serine (most also contain proline andvaline and many contain glycine and isoleucine). In purified form,protamine has been used for decades to neutralize the anticoagulanteffects of heparin. UAs of the invention also include protamine variants(e.g., the +18RGD variant (Wakefield et al, J. Surg. Res. 63:280 (1996))and modified forms of protamine, including those described in PublishedU.S. Application No. 20040121443. Other UAs of the invention includeprotamine fragments, such as those described in U.S. Pat. No. 6,624,141and U.S. Published Application No. 20050101532. UAs of the inventionalso include, generally, peptides that modulate the activity of heparin,other glycosaminoglycans or proteoglycans (see, for example, U.S. Pat.No. 5,919,761). The invention further includes pharmaceuticallyacceptable salts of the above-described UAs, as appropriate, includingsulfate salts.

Proteinaceous UAs of the invention also include DNA and/or RNA reactiveantibodies. For example, anti-nuclear antibodies, such as thoseindicative of lupus erythematosis, Sjögren's syndrome, rheumatoidarthritis, autoimmune hepatitis, scleroderma, polymyositis anddermatomyositis, can be used. Specific examples of antibodies thatrecognize RNA/DNA include those that are described by Kitagawa et al(Mol. Immunol. 19(3):413-20 (1982)), Boguslawski et al (J. Immunol.Methods 89(1):123-30 (1986)), Williamson et al (Proc. Natl. Acad. Sci.98(4):1793-98 (2001)), and Blanco et al (Clin. Exp. Immunol. 86(1):66-70(1991)).

In addition, heterogeneous nuclear ribonucleoproteins (HNRPs) can alsobe used in accordance with the invention. Cationic peptides that bindnucleic acids in a sequence-independent manner are suitable for use. Forexample, a chimeric peptide synthesized by adding nonamer arginineresidues at the carboxy terminus of RVG (to yield RVG-9R) has beendescribed by Kumar et al (Nature 448:39-43 (2007)). Viral proteins thatpackage (e.g., coat) DNA or RNA (e.g., HIV gag protein), and peptidesderived therefrom, can also be used in the present methods.

Cationic lipids can also be used as UAs in accordance with theinvention. Suitable cationic lipids include those described by Morilleet al (Biomaterials 29:3477 (2008)) (e.g., linear poly(ethyleneimine)(PEI), poly(L-lysine) (PLL), poly(amidoamine) (PAMAM) dendrimergeneration 4, chitosan, DOTMA, DOTAP, DMRIE, DOTIM, DOGS, DC-Chol, BGTCand DOPE).

UAs of the invention also include intercalating agents. Examples includeethidium bromide, proflavine, daunomycin, doxorubicin and thalidomide.Porphyrins that bind nucleic acids in a sequence independent manner canalso be used in accordance with the invention. Porphyrins that bindguanine-rich motifs in G-quadruplexes (as described by, for example,Joachimi et al (J. Am. Chem. Soc. 129:3036 (2007)) do not bind in anindependent manner and thus are not within the scope of the invention.

Preferred UAs of the invention include polycationic polymers orpeptides. Preferred polycationic polymers include biocompatible polymers(that is, polymers that do not cause significant undesired physiologicalreactions) that can be either biodegradable or non-biodegradablepolymers or blends or copolymers thereof. Examples of such polymersinclude, but are not limited to, polycationic biodegradablepolyphosphoramidates, polyamines having amine groups on either thepolymer backbone or the polymer side chains, nonpeptide polyamines suchas poly(aminostyrene), poly(aminoacrylate), poly(N-methylaminoacrylate), poly(N-ethylaminoacrylate), poly(N,N-dimethylaminoacrylate), poly(N,N-diethylaminoacrylate), poly(aminomethacrylate),poly(N-methyl amino-methacrylate), poly(N-ethyl aminomethacrylate),poly(N,N-dimethyl aminomethacrylate), poly(N,N-diethylaminomethacrylate), poly(ethyleneimine), polymers of quaternary amines,such as poly(N,N,N-trimethylaminoacrylate chloride),poly(methyacrylamidopropyltrimethyl ammonium chloride); natural orsynthetic polysaccharides such as chitosan, cyclodextrin-containingpolymers, degradable polycations such aspoly[alpha-(4-aminobutyl)-L-glycolic acid] (PAGA); polycationicpolyurethanes, polyethers, polyesters, polyamides, polybrene, etc.Particularly preferred cationic polymers include CDP, CDP-Im, PPA-DPA,PAMAM and HDMBr. (See U.S. Pat. Nos. 7,270,808, 7,166,302, 7,091,192,7,018,609, 6,884,789, 6,509,323, 5,608,015, 5,276,088, 5,855,900, U.S.Published Appln. Nos. 20060263435, 20050256071, 200550136430,20040109888, 20040063654, 20030157030, Davis et al, Current Med. Chem.11(2) 179-197 (2004), and Comprehensive Supramolecular Chemistry vol. 3,J. L. Atwood et al, eds, Pergamon Press (1996).)

UAs of the invention can include compounds of types described in Table1, or derivatives thereof. Several of the compounds described in Table 1contain cationic-NH groups permitting stabilizing charge-chargeinteractions with a phosphodiester backbone. Nucleic acid binding agentsof the invention containing secondary amines can include, for example,5-350 such groups (e.g., 5-300, 5-250, 5-200, 5-100, 5-50, 50-100,50-200, 50-300, 50-350, 100-200, 100-300, 100-350, 200-350, 200-300, or250-350), and can have a molecular weight in the range of, for example,2,000 to 50,000 (e.g., 10,000 to 50,000 or 20,000 to 40,000).

TABLE 1 Compound Abbreviation Molecular structure Remark Poly-L-lysinePLL

1. Commercially available. 2. Carbonyl moiety (—C═O) which could permitadditional stabilization to the complex through hydrogen bonds with DNA.Poly-L-ornithine PLO

1. Commercially available. 2. Carbonyl moiety (—C═O) which could permitadditional stabilization to the complex through hydrogen bonds with DNA.Polyphosphoramidate polymer series PPA-SP PPA-BA PPA- EA PPA-MEA PPA-DMAPPA-DEA PPA-TMA PPA-DPA

1. Polymers with an identical backbone but different side chains rangingfrom primary to quaternary amines. Provide a platform for a systematicstudy. 2. Lower cytotoxicity compared with polyethylenimine (PEI) andpoly-L-lysine (PLL). Polyphosphoramidate diprophylamine- poly ethyleneglycol copolymer PPA-DPA-b- PEG₂₀₀₀

1. a copolymer of PPA-DPA and PEG. Polyethyleneimine PEI

1. Commercially available. 2. PEI with branched structure condenses DNAto a greater extent than linear ones. 3. high cytotoxicity. Ionene e.g.polybrene

1. Commercially available. 2. Have high charge density. Naturalpolyamine H₂N—(CH₂)₄—NH₂ 1. Commercially e.g.H₂N—(CH₂)₃—NH—(CH₂)₄—NH—(CH₂)₃—NH₂ available. PutrescineH₂N—(CH₂)₄—NH—(CH₂)₃—NH₂ 2. The most Spermine extensive work Spermidineon their binding with DNA has been carried out and have remarkableeffects on the DNA condensation. Poly (allylamine) PAL

1. Commercially available. 2. Highly positive charged 3. Low toxicity.Peptide nucleic acid PNA

1. Commercially available. 2. Binding through Watson-crick base pairing,thus binding is typically stronger and more rapid. Poly (porphyrin) orPorphyrin ladder e.g. poly (H₂(p- TAPP poly (por) A- AN))

Poly (2- Methacryloyloxyethyl phosphorylcholine) Dendrimers PMPC

e.g. polyamidoamine dendrimer PAMAM Dendrimer G2

1. Commercially available. 2. Branched spherical shape and a highdensity surface charge. 3. Low cytotoxicity. e.g. polypropyleneiminedendrimer PPI dendrimer

1. A class of amine-terminated polymers, demonstrated to be efficientgene delivery vectors. 2. Low cytotoxicity in a wide range of mammaliancell lines. 3. Unique molecular structures, with defined molecularweight, surface charge and surface functionality. These properties ofdendrimers provide a platform for a systematic study. Partiallydeacetylated Chitin

1. Commercially available. Cyclodextrin grafted branched PEI or linearPEI (α-CD: six sugar ring β-CD: seven sugar ring γ-CD: eight sugar ring)CD-bPEI CD-lPEI

1. Their IC50's are 2-3 orders of magnitude higher than thecorresponding non- cyclodextrin-based polymer. Cyclodextrin ContainingPolymers CDP

CDP-Im

Advantageously, the binding affinity of a UA of the invention for a NAM,expressed in terms of Kd, is in the pM to μM range, preferably, lessthan or equal to 50 nM; expressed in terms of binding constant (K), thebinding affinity is advantageously equal to or greater than 10⁵M⁻¹,preferably, 10⁵M⁻¹ to 10⁸M⁻¹, more preferably, equal to or greater than10⁶M⁻¹. Thus, the binding affinity of the UA can be, for example, about1×10⁵ M⁻¹, 5×10⁵ M⁻¹, 1×10⁶ M⁻¹, 5×10⁶ M⁻¹, 1×10⁷ M⁻¹, 5×10⁷ m⁻¹; orabout 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM. “K” and“Kd” can be determined by methods known in the art, including surfaceplasmon resonance or a real time binding assay such as Biacore.

Certain UAs of the invention, for example, protamine, can be isolatedfrom natural sources (Kossel, The Protamines and Histones (Longmans,N.Y. (1928); Felix et la, Z. Physiol. Chem. 330:205 (1963); Ando et al,Int. J. Prot. Res. 1:221 (1969); Felix, Adv. Prot. Chem. 15:1 (1960).Alternatively, proteinaceous UAs can be produced recombinantly,chemically, or synthetically. UAs described in Table 1 are availablecommercially and/or can be produced using art-recognized techniques.

Standard binding assays, for example, can be used to screen forpreferred UAs of the invention (e.g., using BIACORE and isothermalmicrocalorimetric assays). That is, test compounds (e.g., protaminefragments or variants or modified forms of protamine) can be contactedwith the NAM (e.g., aptamer, etc.) to be targeted under conditionsfavoring binding and a determination made as to whether the testcompound in fact binds the NAM. Test compounds that are found to bindthe NAM can then be analyzed in an appropriate bioassay (which will varydepending on the NAM and its target molecule) to determine if the testcompound can affect the binding of the NAM to its target molecule and/ormodulate (e.g., reverse) the activity of the NAM or modify the NAM andits activity in a functional assay. Test compounds that bind a NAM ofone nucleotide sequence can be screened against a NAM having a differentnucleotide sequence to thereby identify compounds that bind in asequence independent manner.

The UAs of the invention can be used, for example, to reverse theanticoagulant and antithrombotic effects of NAMs (e.g, aptamers, etc.)that target components of the coagulation pathway, particularlyantagonists of the tissue factor (TF)/factor VIIa (FVIIa), factor VIIIa(FVIIIa)/factor IXa (FIXa), factor Va (FVa)/factor Xa (Fxa) enzymecomplexes and platelet receptors such as GPIIb-IIIa and GPVI, factorsinvolved in promoting platelet activation such as Gas6, Von Willebrandfactor, collagen, factors involved in promoting or maintaining fibrinclot formation such as PAI-1 (plasminogen activator inhibitor 1) orcoagulation factor XIIIa (FXIIIa), and additional factors involved inpromoting or preventing fibrin clot formation such as ATIII(anti-thrombin III), thrombin or coagulation factor XIa (FXIa).

UAs of the invention are administered in an amount sufficient tomodulate (e.g., reverse) the NAM activity. Several clinical scenariosexist in which the ability to rapidly reverse the activity of anantithrombotic or anticoagulant NAM is desirable. A first case is whenanticoagulant or antithrombotic treatment leads to hemorrhage, includingintracranial or gastrointestinal hemorrhage. A second case is whenemergency surgery is required for patients who have receivedantithrombotic treatment. This clinical situation arises in a lowpercentage of patients who require emergency coronary artery bypassgrafts while undergoing percutaneous coronary intervention under thecoverage of GPIIb/IIIa inhibitors. Current practice in this situation isto allow for clearance of the compound (for small molecule antagonistssuch as eptifibatide), which may take 2-4 hours, or platelet infusion(for Abciximab treatment). A third case is when an anticoagulant NAM isused during a cardiopulmonary bypass procedure. Bypass patients arepredisposed to post operative bleeding. In each case, acute reversal ofthe anticoagulant effects of a compound via an antidote (e.g., aproteinaceous modulator of the invention) allows for improved, andlikely safer, medical control of the anticoagulant or antithromboticcompound. Similarly, the UAs of the invention can be used when a patienton an anthrombotic is involved in an accident or suffers intracranialhemorrhage and blood loss cannot otherwise be stopped.

UAs of the invention can be used in any of a variety of situations wherecontrol of NAM activity is desired. The targeting of antithrombotic andanticoagulant NAMs is only one example. UAs of the invention can also beused, for example, to modulate (e.g., reverse) the immunosuppressiveeffect of NAMs that target interleukin, for example, in patients subjectto infection. The present UAs can be used, for example, to reverse theimmunostimulatory effects of NAMs that target CTLA4 in patients at riskof developing autoimmunity.

The UAs of the invention can also be used to inhibit NAMs that activatethe immune system. If, for example, a patient goes into systemic shockbecause of over activated immune response due to NAMs binding to immuneactivating cells, UAs of the invention can be used to reverse theeffect.

UAs of the invention can be used to modulate (e.g. reverse) the effectsof NAMs that target receptors involved in the transmission of the nerveimpulse at the neuromuscular junction of skeletal muscle and/orautonomic ganglia (e.g., nicotinic acetylcholine or nicotiniccholinergic receptors). Such NAMs can be made to produce muscularrelaxation or paralysis during anesthesia. Agents that block theactivity of acetylcholine receptors (agents that engender neuromuscularblockade) are commonly used during surgical procedures, and it ispreferred that the patients regain muscular function as soon as possibleafter the surgical procedure is complete to reduce complications andimprove patient turnover in the operating arenas. Therefore, much efforthas been made to generate agents with predictable pharmacokinetics tomatch the duration of the drug activity to the anticipated duration ofthe surgical procedure. Alternatively, UAs of the invention can be usedto provide the desired control of the activity of the neuromuscularblocker, and thus reduce the dependence on the patient's physiology toprovide reversal of the neuromuscular blocking agent.

UAs of the invention can be used to modulate (e.g. reverse) the effectsof NAMs that target growth factors (e.g., PDGF or VEGF). Such NAMs canbe used in the treatment of tumors and in the treatment of inflammatoryproliferative diseases. Since growth factors play systemic roles innormal cell survival and proliferation, NAM treatment can result in abreakdown of healthy tissue if not tightly regulated (e.g., patientsreceiving NAM that target angiopoietin I can be subject tohemorrhaging). UAs of the invention can be used to provide the necessaryregulation.

UAs of the invention can be used to modulate (e.g. reverse) the effectof NAMs that target small molecules, such as glucose. Hypoglycemia canbe avoided in patients receiving glucose-targeted NAMs to regulateglucose uptake using the modulators of the invention. The present UAscan also be used to regulate the activity of NAMs directed againstmembers of the E2F family, certain of which are pro-proliferative,certain of which are repressive. The UAs of the invention can be used to“turn off” such NAMs at desired points in the cell cycle.

UAs of the invention can also be used to reverse the binding of NAMsbearing radioactive, cytotoxic or other therapeutic moieties to targettissue (e.g., neoplastic tissue) and thereby, for example, facilitateclearance of such a moiety from a patient's system and thereby controlor limit patient exposure.

The UAs of the invention can also be used to reverse the binding of NAMslabeled with detectable moieties (used, for example, in imaging (e.g.,PET or CT) or cell isolation or sorting) to target cells or tissues(see, generally, Hicke et al, J. Clin. Invest.

106:923 (2000); Ringquist et al, Cytometry 33:394 (1998)). This reversalcan be used, for example, to expedite differential imaging of thedetectable moiety. For example, an aptamer that bears a detectable labeland that recognizes, for instance, cells of a tumor or a thrombosis, canbe administered to a patient under conditions such that the labeledaptamer binds to the tumor or clot. Administration of a UA of theinvention can rapidly reverse the binding of the labeled aptamer to itstarget. The ability to effect this rapid reversal makes possibledifferential real time imaging of the tumor or clot.

The UAs of the invention can also be used in in vitro settings tomodulate (e.g., inhibit) the effect of a NAM (e.g., aptamer, etc.) on atarget molecule. For example, a UA of the invention can be used tomodulate (e.g., reverse) the effect of a NAM on a particular targetmolecule, present in a mixture of target molecules.

The UAs of the invention (including nucleic acid binding polymersincorporated into microparticles or nanoparticles or beads), orpharmaceutically acceptable salts thereof, can be formulated intopharmaceutical compositions that can include, in addition to the UA, apharmaceutically acceptable carrier, diluent or excipient.“Pharmaceutically acceptable” with respect to a component, such as asalt, carrier, excipient or diluent of a composition according to thepresent invention is a component that is compatible with the otheringredients of the composition, in that it can be combined with the UAof the present invention without eliminating the biological activity ofthe UA, and is suitable for use with subjects as provided herein withoutundue adverse side effects (such as toxicity, irritation, and allergicresponse). Side effects are “undue” when their risk outweighs thebenefit provided by the composition comprising the UA. Examples of suchcarriers include, but are not limited to, aqueous solutions, aqueous ornon-aqueous solvents, suspensions, emulsions, gels, pastes, and thelike. As known to those skilled in the art, a suitable pharmaceuticallyacceptable carrier can comprise one or more substances, including butnot limited to, water, buffered water, medical parenteral vehicles,saline, 0.3% glycine, aqueous alcohols, isotonic aqueous buffer; and canfurther include one or more substances such as water-soluble polymer,glycerol, polyethylene glycol, glycerin, oils, salts such as sodium,potassium, magnesium and ammonium, phosphonates, carbonate esters, fattyacids, saccharides, polysaccharides, glycoproteins (for enhancedstability), excipients, and preservatives and/or stabilizers (toincrease shelf-life or as necessary and suitable for manufacture anddistribution of the composition).

The precise nature of the compositions of the invention will depend, atleast in part, on the nature of the UA and the route of administration.Optimum dosing regimens can be readily established by one skilled in theart and can vary with the UA, the NAM, the patient and the effectsought. Generally, the NAM and UA can be administered orally,transdermally, IV, IM, IP, SC, or topically, as appropriate.

The amount of UA administered is sufficient to inhibit or reversebinding of a NAM to its target. Preferably, at least 50% of the NAMs areinhibited from binding target molecules or, for those NAMs bound totarget molecules, are interrupted and freed from binding (“reversal”)their target molecules, as compared to binding in the absence of the UA.

Furthermore, because the antidote activity is durable, once the desiredlevel of modulation of the NAM by the antidote is achieved, infusion ofthe antidote can be terminated, allowing residual antidote to clear thehuman or animal. This allows for subsequent re-treatment of the human ornon-human animal (e.g., farm animal such as a cow, pig, horse, goat orsheep, or companion animal such as a dog or cat) with the NAM as needed.

Proteinaceous UAs of the invention can also be produced in vivofollowing administration of a construct comprising a sequence encodingthe proteinaceous UA (Harrison, Blood Rev. 19(2):111-23 (2005)).

Certain aspects of the invention are described in greater detail in thenon-limiting Examples that follows. Incorporated by reference is thefollowing citation that describes APTT and other clotting assays: Quinnet al, J. Clin. Lab. Sci. 13(4):229-238 (2000). This review describesthe properties and biochemistry of various clotting assays includingAPTT, PT and thrombin time assays, and their use in diagnosingcoagulopathies.

Example I

Millions of individuals have received protamine, a group of positivelycharged proteins, to reverse the blood thinning effects of heparin,particularly following cardiopulmonary bypass surgery. A crystalstructure study on an RNA aptamer that binds human thrombin showed thatthe aptamer bound to thrombin in a similar location as heparin. To testthe hypothesis that aptamers may have other properties similar toheparin, studies were undertaken to determine if aptamer activity couldbe neutralized with protamine, since heparin activity can be reversedusing protamine.

To test this possibility, human plasma was anticoagulated with twodifferent aptamers, one to human factor IXa (termed CH-9.3t (37 nt, 2′fluoropyrimidine modified ssRNA molecule with a cholesterol moiety onthe 5′ end, 3′idt) 9.3t (same as CH-9.3t except no carrier)) and asecond one to human factor Xa (termed 11f7t (37 nt long, 2′fluoropyrimidine modified RNA molecule)).

As shown in FIG. 1, addition of the aptamers to human plasma (150 nM forCh9.3t and 250 nM 11f7t) significantly increased the clotting time asmeasured in an aPTT assay. In the case of the factor IXa aptamer,clotting time increased from a baseline of approximately 34 seconds toapproximately 87 seconds (FIG. 1A). Addition of protamine (2.5 μg) tothe plasma after it had been anticoagulated with the factor IXa aptamerreturned the clotting time to normal within 5 minutes followingaddition. This reversal was maintained for at least an hour. Similarly,the factor Xa aptamer (250 nM) increased the clotting time of normalhuman plasma in the aPPT assay from approximately 28 seconds toapproximately 97 seconds. As shown in FIG. 1B, administration ofprotamine (2.5 μg) totally reversed the activity of the factor Xaaptamer within 5 minutes. (See also FIG. 2.)

Example II

Table 2 includes a summary of data resulting from UA vs aptamerexperiments (see Example I above (Bi-9.3t (37 nt, 2′ fluoropyrimidinemodified ssRNA with biotin on the 5′ end)).

TABLE 2 Universal Antidote vs Aptamer Experiments Aptamer APTT clotting(150 nM) Antidote time (sec) Reversal Comments 0 0 34.5 These areabbreviated results. 9.3t-Bi 0 86.6 All the concentrations areoptimized. 0 Protamine (2.5 ug/well) 24 9.3t-Bi Protamine (2.5 ug/well)30.3 Yes 0 0 30.5 9.3t-Bi 0 85.2 0 Poly-L Lysine (5 KDa)(1.3 ug/well)30.7 9.3t-Bi Poly-L Lysine (5 KDa) (1.3 ug/well) 81 No 0 0 33.5 9.3t-Bi0 83.1 0 Poly-L Lysine (300 KDa)(1.3 ug/well) 30.7 9.3t-Bi Poly-L Lysine(300 KDa) (1.3 ug/well) 39.4 Yes 0 0 31.3 9.3t-Bi 0 76.1 0 Poly-L Lysine(360 KDa)(1.3 ug/well) 37.9 9.3t-Bi Poly-L Lysine (360 KDa) (1.3ug/well) 43.9 Yes 0 0 36.2 9.3t-Bi 0 91.1 0 Spermine (1.3 ug/well) 63.89.3t-Bi Spermine (1.3 ug/well) 72.7 NO Did not work b/w 1.3 ug/well-10ug/well 0 0 28.8 9.3t-Bi 0 78.7 0 PPA-DPA 8 KDa (65 ug/well) 58.29.3t-Bi PPA-DPA 8 KDa (1.3 ug/well) 63.9 NO Did not work b/w 1.3ug/well-65 ug/well 0 0 27.9 9.3t-Bi 0 69.9 0 PPA-DPA 30 KDa (1.3ug/well) 28.3 9.3t-Bi PPA-DPA 30 KDa (1.3 ug/well) 36.2 Yes 0 0 27.59.3t-Bi 0 75.9 0 Peg-b-PPA (1.3 ug/well) 24.8 9.3t-Bi Peg-b-PPA (1.3ug/well) 58.8 No Did not work b/w 1.3 ug/well-65 ug/well

Example III

The Platelet Function Analyzer (PFA-100). The PFA-100 is a bench topinstrument that uses whole blood and simulates platelet function underhigh shear stress conditions. In this experiment, disposable cartridgescoated with Collagen/ADP were filled with 840 microliters of whole humanblood (collected in 10 ml sodium heparin tubes) and placed into thePFA-100. The standard test protocol is followed and each dilution pointis done in duplicates. For antidote experiments, 50 nM of vWF aptamerR9.3 was incubated with the whole blood for 5 minutes followed by theaddition of antidote. The blood was than placed in PFA-100 and the testwas run.

During the test, blood in the cartridge is aspirated under constantnegative pressure from the reservoir, through a capillary, passing amicroscopic aperture cut into the membrane. The shear stress rate duringthis process reaches 5000-6000 s⁻¹ and along with the plateletactivators (i.e. Collagen/ADP) present on the membrane, initiatesplatelet activation, adhesion and aggregation. These processes cause theformation of a platelet plug on the microscopic aperture and blood flowthrough the capillary ceases. The platelet function is measured as thetime it takes to form the aperture occlusion. Although the PFA-100 issensitive to many variables that affect platelet function, a number ofstudies revealed that it is most sensitive to certain platelet receptordefects (mainly GPIb-IX-V and GP IIbIIIa) and VWF defects. (See FIG. 3.)

Example IV Experimental Details

Swine (2.5-3.5 kg) were randomly assigned to treatment groups. For allgroups, anesthesia was induced by intramuscular injection of ketamine(22 mg/kg) and acepromazine (1.1 mg/kg). A catheter was then placed inthe ear vein, through which anesthesia was maintained with fentanyl,first with a 100 μg/kg bolus, and then with a continuous infusion of 60μg·kg⁻¹·h⁻¹. The swine were then intubated and mechanically ventilated.Following placement of the esophageal or rectal temperature probe andSPO₂ _(—) monitor, the femoral artery and vein were cannulated. Thearterial line was used as a means to continuously monitor mean arterialblood pressure and heart rate, as well as removal of blood samples forevaluation of ACT. After determining baseline ACT values, the venousline was used to administer the Factor IXa aptamer (0.5 mg/kg). Bloodsamples were then drawn from the arterial line at 5, 15, and 30 minutespost aptamer administration. The ACT values were measured by usingHemochron Jr. Signature Microcoagulation System (ITC, Edison N.J.). Forexperiments involving antidote administration, 40 mg protamine (10mg/mL) was given over five minutes via the femoral vein catheter at 30minutes post aptamer injection. For all animals, subsequent bloodsamples were at taken at 35, 40, 55, 60, 75, and 90 minutes post aptameradministration. All data points are done in duplicate per animal. At theclosure of the experiment, swine were euthanized with Euthasol (175mg/kg) via femoral vein.

All animals received humane treatment in accordance with the Guide forthe Care and Use of Laboratory Animals published by the National isInstitutes of Health, as approved by the Duke University Animal Care andUse Committee.

Results

Immediate anticoagulation and prompt reversal is necessary duringseveral cardiovascular procedures, the most common being cardiopulmonarybypass (CPB), as employed during coronary artery bypass grafting (CABG).Without potent anticoagulation, blood clots would form and blood wouldnot circulate in an extracorporeal circuit. However, following theprocedure, these values must return to pretreatment levels to preventmassive hemorrhage from the surgical site. After successfullydetermining the ability of protamine to reverse the activity of theFactor IXa and Factor Xa aptamers for at least 60 minutes in vitro usingAPTT, the ability of protamine to reverse aptamer anticoagulation invivo was examined. As shown in FIG. 4A, pigs were successfullyanticoagulated with 0.5 mg/kg of the Factor IXa aptamer, as demonstratedby an immediate increase in the ACT from 105 s. to approximately 170 s.When no antidote was administered, the level of anticoagulationgradually decreased in accordance with the 90-minute half-life of themolecule (Rusconi et al, Nat. Biotechnol. 22(1):1423-1428 (2004)).However, following administration of 10 mg/kg of protamine (approx. 40mg per animal), clotting times as measured by ACT quickly returned topretreatment baseline levels within five minutes, indicating completereversal of anticoagulation (FIG. 4B). In addition, this reversal wassustained for the duration of the experiment (at least one hour); allACT values attained post reversal are below the level needed foradequate anticoagulation (FIG. 4C). Therefore, a bolus injection of theFactor IXa aptamer resulted in immediate anticoagulation that wassuccessfully reversed with protamine.

Example V

In order to reverse the aptamer function, an antidote has to be able tocompete with the target protein by binding to the aptamer with highaffinity. Binding affinities of aptamer 9.3t with a number of polymersand polycationic molecules have been studied by isothermal titrationcalorimetry (ITC). In the measurements, UA solution was titrated intothe aptamer 9.3t by a computer-controlled microsyringe at 298K in PBS.Binding constants and some thermodynamic parameters are summarized inTable 3. Among the chosen polymers, PAMAM dendrimer, PPA-DPA 30k and PLLdemonstrated significant affinity with aptamer 9.3t; whereas, thebinding constant of aptamer 9.3t to PPA-DPA 8k is much lower, at8.47×10⁵ M⁻¹. No significant binding was observed for the naturalpolyamines, spermine and spermidine. These results are consistent withthe in vitro and in vivo studies which showed that having a strongaffinity towards aptamers is a primary criterion as a UA.

TABLE 3 Binding TΔS Potential constant ΔG ΔH (kJ Host Antidotes (M⁻¹)(kJ mol⁻¹) (kJ mol⁻¹) mol⁻¹) Aptamer Polybrene 1.6 × 10⁵ −29.6 +71.3+101.0 Ch-9.3t PAMAM 1.7 × 10⁶ −35.5 −198.5 +163.0 Spermine 8.8 × 10³−22.5 −1.18 +21.3 PPA-DPA 8k 2.7 × 10⁵ −31.0 +24.7 +55.7 PPA-DPA 30k 1.3× 10⁷ −40.5 +138.24 +178.8

The isothermal calorimetry measurements (ITC) were conducted by using athermostatic and fully computer-operated MCS-ITC calorimeter fromMicroCal, LLC. Aliquots of 10 μL were titrated into the calorimetriccell every 5 min over a 2 hours period at 298K. A blank run was carriedout for each system studied where the titrant was titrated into a cellcontaining only PBS to allow corrections for the heat effects due todilution to be made. Data analysis was performed using the customizedITC module of the Origin 5.0 software package and a least squaresfitting procedure to fit the data to the appropriate binding model.

Example VI Experimental Details Clotting Assays

Activated partial thromboplastin time (APTT) assays were performed usinga model ST4 mechanical coagulometer (Diagnostica Stago Inc.). Poolednormal human plasma (50 μl) (George King Biomedical) was incubated at37° C. for 5 min followed by the addition of platelin reagent (50 μl)(Trinity Biotech) and aptamer (in wash buffer) (5 μl) or wash bufferalone, and incubated for 5 min at 37° C. Antidote molecule or buffer (5μl) was than added and incubated at 37° C. for 5 min followed by theaddition of 25 mM CaCl₂ (50 μl) to initiate the clotting reaction(Rusconi et al, Nature 419:90 (2002)). Data is shown as the change inclot time. All reactions were performed in triplicate.

Platelet Function Assays

The Platelet Function Analyzer, PFA-100 (Dade Behring, Deerfield, Ill.),measures platelet function in terms of clot formation time. In thisassay, collagen/ADP cartridges were used to activate the platelets. 800μl of whole blood was mixed with aptamer in platelet binding buffer (40μl) (150 mM NaCl; 20 mM Hepes pH: 7.4; 5 mM KC1; 1 mM MgCl₂ and 1 mMCaCl₂) and incubated for 5 min at room temperature. Antidote molecule orplatelet buffer (40 μl) was than added and incubated for 5 min at roomtemperature. This mixture was then added to a collagen/ADP cartridge andtested for its closing time (Harrison, Blood Rev. 19:111 (2005)). Themaximum closing time of the PFA-100 is 300 seconds. Data is shown as thechange in closing time. All reactions were performed in triplicate.

Isothermal Calorimetry

The isothermal calorimetry measurements (ITC) were conducted by using athermostatic and fully computer-operated MCS-ITC calorimeter fromMicroCal, LLC. Aliquots of 10 μl were titrated into the calorimetriccell every 5 min over a 2 hour period at 298K. A blank run was carriedout for each system studied where the titrant was titrated into a cellcontaining only PBS to allow corrections for the heat effects due todilution to be made. Data analysis was performed using the customizedITC module of the Origin 5.0 software package and a least squaresfitting procedure to fit the data to the two-sites binding model.

In Vitro Assessment of CDP/9.3t Interaction by Gel Electrophoresis andDynamic Light Scattering (DLS)

Gel electrophoresis was performed by adding 5 μl volume of 0.01 mg/mL9.3t in 1×PBS, an equal volume of CDP in 1×PBS. This was performed sixtimes in parallel using CDP solutions of various concentrations to giveresulting solutions having CDP/9.3t ratios (w/w) of 0/1 (lane 2), 2/1(lane 3), 4/1 (lane 4), 6/1 (lane 5), 8/1 (lane 6), and 10/1 (lane 7).To each of these 10 μl solutions, 2 μl of nucleic acid loading bufferwas added, and 10 μl of each resulting solution was loaded per well of a15% polyacrylamide/TBE gel and electrophoresed (1 h, 100 V). The gel wasthen incubated in 1×TBE buffer containing 0.5 μg/mL ethidium bromide for30 min and visualized. CDP alone (0.0335 mg/mL in 1×PBS), 9.3t alone(0.005 mg/mL in 1×PBS), and CDP/9.3t (0.0335 mg/mL CDP, 0.005 mg/mL9.3t; 6.7/1 w/w in 1×PBS) were analyzed by dynamic light scattering(DLS) using a ZetaPALS particle size analyzer. Three consecutive3-minute runs were performed for each sample; the effective diameter andaverage particle count rate were determined.

Analysis of CDP-Im-siRNA Complex Formation In Vivo.

Four female BALB/c mice received a single tail-vein injection of naked,non-chemically-modified siRNA. One minute after this injection, two ofthe mice received a separate tail-vein injection of CDP-Im along withtwo other typical nanoparticle components, AD-PEG and AD-PEG-Tf (Heidelet al, Proc. Natl. Acad. Sci. USA 104:5715 (2007)). Two minutes later,blood was collected from all mice and centrifuged to separate serum.Serum samples were then electrophoresed using a 156 TBE polyacrylamidegel and visualized using ethidium bromide.

Swine Systemic Anticoagulation and Reversal Study

Swine (2.5-3.5 kg) were randomly assigned to treatment groups. For allgroups, anesthesia was induced by intramuscular injection of ketamine(22 mg/kg) and acepromazine (1.1 mg/kg). A catheter was then placed inthe ear vein, through which anesthesia was maintained with fentanyl,first with a 100 μg/kg bolus, and then with a continuous infusion of 60μg·kg⁻¹·h⁻¹. The swine were then intubated and mechanically ventilated.Following placement of the esophageal or rectal temperature probe andSP0₂ monitor, the femoral artery and vein were cannulated. The arterialline was used as a means to continuously monitor mean arterial bloodpressure and heart rate, as well as removal of blood samples forevaluation of ACT. After determining baseline ACT values, the venousline was used to administer the Factor IXa aptamer (0.5 mg/kg). Bloodsamples were then drawn from the arterial line at 5, 15, and 30 minutespost aptamer administration. The ACT values were measured by usingHemochron Jr. Signature Microcoagulation System (ITC, Edison N.J.). Forexperiments involving antidote administration, 40 mg protamine (10mg/mL) or 10 mg CDP (10 mg/ml) was given over five minutes via thefemoral vein catheter at 30 minutes post aptamer injection. For allanimals, subsequent blood samples were at taken at 35, 40, 55, 60, 75,and 90 minutes post aptamer administration. All data points are done induplicate per animal. At the closure of the experiment, swine wereeuthanized with Euthasol (175 mg/kg) via femoral vein.

All animals received humane treatment in accordance with the Guide forthe Care and Use of Laboratory Animals published by the NationalInstitutes of Health, as approved by the Duke University Animal Care andUse Committee.

Results

Two aptamers selected to different targets (thrombin and VEGF) have beenshown to interact with the heparin binding domain (HBD) on their targetproteins (Lee et al, Proc. Natl. Acad. Sci. USA 102:18902 (2005), Whiteet al, Mol. Ther. 4:567 (2001)). An initial investigation was made as towhether protamine, the antidote for heparin, could reverse the activityof two aptamers that target coagulation factors IXa (aptamer 9.3t) andXa (aptamer 11F7T). The activated partial thromboplastin time (APTT)assay is used for diagnosing coagulation factor anomalies and also formonitoring anticoagulation therapy. It has been demonstrated previouslythat the APTT assay could be utilized to monitor the anticoagulanteffects of both FIXa and FXa aptamers (Rusconi et al, Nature 419:90(2002), Rusconi et al, Nat. Biotechnol. 22:1423 (2004)) (unpublishedresults). These aptamers have significantly different primary sequencesand secondary structures (as predicted by mFold, FIG. 5A) (Zuker,Nucleic Acids Res. 31:3406 (2003)) and target different proteins in thecoagulation cascade. As shown in FIG. 5B, aptamer 9.3t is a potentanticoagulant. However, addition of protamine neutralized theanticoagulant effects of this aptamer within 5 minutes (FIG. 5B).Similarly, aptamer 11F7T is a potent anticoagulant agent and protaminecan also neutralize the activity of this aptamer (FIG. 5C). In bothcases, 2.5 μg of protamine was able to totally reverse the aptamers'activity in an APTT clotting assay (FIG. 5D) at a 2 fold lowerconcentration than is routinely used to reverse heparin's anticoagulantactivity. Moreover, protamine was able to reverse the anticoagulantactivity of the two aptamers simultaneously (FIG. 5E) and such reversalwas maintained for at least an hour.

Although protamine is routinely used to reverse the activity of heparinfollowing cardiopulmonary bypass surgery, protamine administration isassociated with several side effects, including increased pulmonaryartery pressure, decreased systolic and diastolic blood pressure,impaired myocardial oxygen consumption, and reduced cardiac output,heart rate, and systemic vascular resistance (Nimjee et al, Mol. Ther.14:408 (2006), Hird et al, Circulation 92:II1433 (1995), Porsche et al,Heart Lung 28:418 (1999), Shigeta et al, J. Thorac. Cardiovasc. Surg.118:354 (1999), Welsby et al, Anesthesiology 102:308 (2005)). Therefore,the decision was made to identify other agents that could rapidlyreverse the activity of aptamers. A number of nucleic acid bindingpolymers were screened for their ability to act as antidotes for aptamer9.3t (Table 4). As shown in FIG. 6A, several of the polymers were ableto completely reverse the activity of the aptamer completely within 5minutes. To better understand why certain polymers were more effectivethan others, the interactions of the different polymers with aptamer9.3t were measured by isothermal titration calorimetry (ITC). A two-sitemodel was used to interpret the ITC data for the interactions betweenthe cationic polymers and aptamer 9.3t. Binding constants and somethermodynamic parameters are summarized in Table 5. All the interactionsare entropy driven (as expected) except for with PAMAM, and most arealso enthalpy driven. Although no conclusions can be drawn about themechanisms of the interactions, the trends of binding strength, in thetwo-site model, are consistent with the results from the APTT screening.Protamine, CDP, CDP-Im, PPA-DPA and PAMAM demonstrated significant andsimilar affinities for aptamer 9.3t; whereas the binding constants ofpolybrene and spermine are orders of magnitude lower. Thus, a directcorrelation exists between the polymer's affinity for the aptamer andits potency as an antidote.

Since CDP has high binding affinity for the aptamer and is known to havea low toxicity, this polymer was tested in time course experiments(Gonzalez et al, Bioconjug. Chem. 10:1068 (1999)). Similar to protamine,CDP could rapidly and durably reverse the activity of these two distinctanticoagulant aptamers (9.3t and 11F7T) in vitro (FIGS. 6B-6D). Next, anexamination was made of CDP's ability to reverse the activity of fouradditional aptamers that target factors II, IX, X and VII. As shown inFIGS. 6E and 6F, CDP can rapidly reverse the activity of each of theseaptamers.

TABLE 4 Definitions and molecular structures of the selected potentialantidote. Polymer Abbreviation Molecular structure β-cyclodextrin-containing polycation CDP

β-cyclodextrin- containing polycation (imidazole-containing variant)CDP-Im

Polyphosphoramidate polymer (8kDa. 30kDa) PPA-DPA 8k, PPA-DPA 30k

Polyamidoamine dendrimer, 1,4- diaminobutane core, G3 PAMAM[NH₂—(CH₂)₄—NH₂]:(G = 3); dendri PAMAM(NH₂)₃₂ Polybrene

Spermine NH₂—(CH₂)₃—NH—(CH₂)₄—NH—(CH₂)₃—NH₂

TABLE 5 Binding constants and thermodynamic parameters of (a) the firststage binding and (b) the second stage binding, between the selectedpotential antidotes and aptamer 9.3t. a. ΔG ΔH TΔS Polymer K1 (M⁻¹) (kJmol⁻¹) (kJ mol⁻¹) (kJ mol⁻¹ K) protamine 3.09E+07 −42.73 −37.57 5.16 CDP2.63E+07 −42.33 −27.93 14.41 CDP-lm 3.69E+08 −48.87 −44.52 4.35 PPA 30k6.71E+08 −50.35 100.58 150.94 PPA 8K 2.46E+07 −42.16 24.84 67.00 PAMAM7.37E+08 −50.59 −225.22 −174.64 polybrene 9.23E+06 −39.74 63.60 103.33spermine 1.63E+06 −35.43 −1.01 34.42 b ΔG ΔH TΔS Polymer K2 (M⁻¹) (kJmol⁻¹) (kJ mol⁻¹) (kJ mol⁻¹ K) CDP 1.20E+07 −40.39 −6.89 33.50 CDP-lm4.54E+07 −43.68 −26.85 16.83 PPA 30k 1.03E+07 −40.00 134.93 174.94 PPA8K 2.28E+06 −36.27 4.20 40.47 PAMAM 3.69E+07 −43.17 −68.24 −25.07polybrene 8.03E+05 −33.68 3.04 36.72 spermine 1.26E+05 −29.10 −0.3028.80

CDP and PPA-DPA were next tested for their ability to neutralize theantiplatelet effects of VWF aptamer 9.3 and VWF aptamer 9.14 (FIG. 5A)in a platelet function assay (PFA-100) (Oney et al, Oligonucleotides17:265 (2007)). VWF aptamers 9.3 and 9.14 have no sequence or structuralsimilarity to the previously tested aptamers and both can inhibitplatelet function in while blood (FIGS. 7A and 7B). Addition of eitherCDP or PPA-DPA and resulted in rapid reversal of VWF aptamer 9.3antiplatelet activity (FIGS. 7A and 7B) with CDP achieving completereversal at an order of magnitude lower amount than PPA-DPA. Moreover,CDP was able to rapidly reverse the activity of VWF aptamer 9.14 at thissame concentration (FIG. 7C). These experiments further demonstrate thatCDP and PPA-DPA can act as sequence-independent antidotes for aptamers.Furthermore, these results point to the broad applicability of thisapproach since the antidotes work in both plasma and whole blood againsteight different aptamers.

Next, studies were undertaken to determine whether such UAs were able toreverse aptamer activity in vivo. Results from in vitro experiments (gelelectrophoresis and dynamic light scattering) using the sameconcentrations as anticipated for use in animals demonstrated that CDPis able to bind to the aptamer and form a composite entity (FIG. 9) andit was observed that CDP-Im formed a complex with siRNA whensequentially injected into mice (FIG. 11). Therefore, the activity ofthe UAs was evaluated in a swine anticoagulation model. As seen in FIG.8A, pigs (n=5) were anticoagulated by the FIXa aptamer (Ch-9.3t) (0.5mg/kg) that had been modified with a cholesterol at its 5′ end toimprove its circulating half life (Rusconi et al, Nat. Biotechnol.22:1423 (2004)). An immediate increase was observed in the activatedclotting time (ACT) (from 105+/−5 seconds to 150+/−5 seconds) for thetreated animals. When no antidote was administered, the level ofanticoagulation only gradually decreased over the 90 minute time frameof the experiment (FIG. 8A). However, administration of protamine (10mg/kg) resulted in a total reversal of the anticoagulant effect withinfive minutes (n=5) (FIG. 8B). In addition, this reversal was sustainedfor the remainder of the experiment, 60 minutes (FIG. 8B). Similarly,CDP (n=5) (2.5 mg/kg) was also able to rapidly and durably reverse theactivity of this aptamer in vivo (FIG. 8C). Furthermore, no toxicitieswere observed following administration of these antidotes during theexperiment (FIG. 10). All vital signs stayed within error of their baseline levels with the exception that protamine induced a mild hypotensionand CDP a mild hypertension (<15% change; FIGS. 10D and 10E). Theseresults demonstrate that both protamine and CDP can act as antidotes foraptamers in vivo.

Between 1998 and 2005, the number of serious adverse drugs eventsreported to the FDA increased 2.6-fold, and fatal adverse eventsincreased 2.7-fold to 15,107 events in 2005 (Moore et al, Arch. Intern.Med. 167:1752 (2007); Lazarou et al, JAMA 279:1200 (1998)). Therefore, apressing medical need exists to develop safer and more controllabletherapeutic strategies. Unfortunately, it has been both technicallychallenging and cost prohibitive to develop antidote molecules tocounteract the side effects of most medicines. However, characteristicsunique to oligonucleotides can be used to design UAs that can sequesteraptamers and reverse their activity regardless of the aptamer's primarysequence and folded structure. Initial studies demonstrated thatprotamine, a commonly used and inexpensive heparin reversal agent withwell known side effects can be utilized as an antidote for multipleaptamers (Carr et al, J. Cardiovasc. Surg. (Torino) 40:659 (1999),Stanker et al, Mol. Immunol. 30:1633 (1993)). Furthermore, theobservation that protamine can neutralize aptamer activity indicatesthat protamine should be used with caution in patients being treatedwith oligonucleotide-based drugs since protamine may unintentionallyreverse the activity.

In the quest to find UAs with more favorable characteristics, such aslow toxicity, a number of polymeric gene carriers were tested for theirability to reverse the activity of aptamers. The field of nonviral genetherapy has stimulated the synthesis of many polymers for the deliveryof plasmid DNA and siRNA. Rigidity, hydrophobicity/hydrophilicity,charge density, biodegradability, and molecular weight of the polymerchain are all parameters that can be adjusted to achieve an optimalcomplexation with oligonucleotides (Davis et al, Curr. Med. Chem. 11:179(2004), Mao and Leong, Adv. Genet. 53:275 (2005)). Moreover, Joachimi etal. demonstrated that a small cationic porphyrin could act as anantidote to a G-quartet containing thrombin aptamer (Joachimi et al, J.Am. Chem. Soc. 129:3036 (2007)). Therefore, in the study describedherein, a number of DNA or siRNA delivery polymers were screened. It wasdemonstrated that several of them can reverse the activity of multipleaptamers in vitro and that CDP can also rapidly reverse the activity ofan anticoagulant aptamer in large animals.

Previously, a strategy was developed to reverse the activity of aptamersusing Watson-Crick base pairing rules to create a customized antidoteoligonucleotide for each aptamer (Rusconi et al, Nature 419:90 (2002),Rusconi et al, Nat. Biotechnol. 22:1423 (2004)). This customization isvery costly since for each aptamer a new antidote oligonucleotide has tobe developed, tested and manufactured. Another concern with thisapproach is that when antidote oligonucleotides bind to aptamers, adouble stranded RNA is formed. This double stranded RNA complex maystimulate the innate immune system (Kleinman et al, Nature 452:591-597(2008), Epub 2008 Mar. 26; Schroder and Bourie, Trends Immunol. 26:462(2005)). Although both the RNA aptamer and the antidote oligonucleotideare often comprised of modified oligonucleotides (i.e. 2′-F and 2′-OmeRNA) a potential danger still exists that these molecules may activateTLR-3 receptors as short 2′Ome siRNA duplexes have been reported toinduce such effects (Kleinman et al, Nature 452:591-597 (2008); Epub2008 Mar. 26). Finally, because most indications will not require anantidote for the reversal of drug action 100% of the time, the addedcost of developing a customized antidote is difficult to justify formost drugs that usually are safe but are associated with relatively rarebut serious side effects. Thus, it is believed that the UA approachdescribed herein will be more broadly applicable than the previouscustomized antidote oligonucleotide previously.

As with any new therapeutic agent, the safety of the UA molecules willhave to be tested in the clinic. Recent studies evaluating the toxicityof CDP-Im in non-human primates demonstrate that such compounds have afavorable safety profile (Heidel et al, Proc. Natl. Acad. Sci. USA104:5715 (2007)) leading Calando Pharmaceuticals Inc. to receive FDAapproval for a first in human study using this compound to deliversiRNAs in cancer patients. Thus, there is reason to be optimistic thatthe UA strategy describe herein can be rapidly translated into theclinic.

1. A method of modulating the activity of a nucleic acid molecule (NAM)that binds to a target molecule and elicits a pharmacological effect,said method comprising contacting said NAM with a universal antidote(UA) under conditions such that said UA binds to said NAM and modifiesthe interaction between said NAM and said target.
 2. The methodaccording to claim 1 wherein said method is a method of reversing orinhibiting the activity of said NAM.
 3. The method according to claim 1wherein said NAM is an aptamer, a siRNA, a microRNA, a ribozyme or anantagomir.
 4. The method according to claim 3 wherein said NAM is anaptamer.
 5. The method according to claim 1 wherein said target moleculeis a peptide, protein, glycoprotein, polysaccharide or nucleic acid. 6.The method according to claim 5 wherein said target molecule is anenzyme, hormone, receptor, adhesion molecule, metabolite, or cofactor.7. The method according to claim 5 wherein said target molecule isfactor VIIa, factor IXa, factor Xa, factor XIa, thrombin or protein C.8. The method according to claim 1 wherein said UA is a pharmaceuticallyacceptable positively charged protein, lipid, or natural syntheticpolymer, or pharmaceutically acceptable salt thereof.
 9. The methodaccording to claim 8 wherein said UA is protamine or fragment thereof.10. The method according to claim 1 wherein said UA reverses an immunesystem activating effect of said NAM.
 11. The method according to claim2, wherein said UA reverses an anticoagulant and antithrombic effect ofsaid NAM.
 12. The method according to claim 2 wherein said UA reversesan immunosuppressive effect of said NAM.
 13. The method according toclaim 2 wherein said UA reverses an acetylcholine receptor blockingactivity of said NAM.
 14. The method according to claim 1 wherein saidcontacting is effected in vivo.
 15. The method according to claim 14wherein said UA is produced in vivo following administration to apatient in need thereof a construct comprising a nucleic acid sequenceencoding said UA.
 16. The method according to claim 15 wherein said UAprotamine and said NAM is an aptamer to human factor IXa or human factorXa.
 17. The method according to claim 14 wherein said contacting iseffected in a mammal.
 18. The method according to claim 17 wherein saidmammal is a human.
 19. A method of screening for a candidate UAcomprising contacting a test compound with a NAM and determining whethersaid test compound binds to said NAM, wherein a test compound that bindsto said NAM is a candidate UA.